Fluorescent probes are functional molecules which are substantially non° fluorescent in the absence of a target substance, and become fluorescent after a reaction with the target substance. Since fluorescent probes achieve measurement of a target substance at an extremely low concentration under mild conditions such as neutral pH and physiological temperature, and enable highly sensitive imaging of a target substance existing in living tissues or organs, they are widely applied as probes for measurement of nitric oxide, reactive oxygen species, metal ions, and the like. As fluorescent probes for measuring a protease, the following probes have been proposed (Biochemical Journal, 201, pp. 367-372, 1982).
Xanthene dyes such as fluorescein and rhodamine are used as fundamental skeletons of fluorescent probes. Xanthene dyes are strongly fluorescent when they have an open ring structure. Whilst, when they have a closed ring structure in which a lactone ring is formed, the conjugation of the fluorophore is cleaved, and therefore they are substantially non-fluorescent. For applying this phenomenon to off/on control of fluorescence, fluorescent probes have been designed which react with a target substance so that structural change from the closed ring structure to the open ring structure is induced.
For example, the following diacyl type fluorescent probe for measuring a protease using the rhodamine structure is known. This diacyl type fluorescent probe becomes a weakly fluorescent compound having an open ring structure due to hydrolysis of the acyl group at one side by a protease, and when the other acyl group is further hydrolyzed, strongly fluorescent diaminorhodamine is generated (Biochemistry, 38, pp. 13906-13911, 1999).

However, this fluorescent probe must be hydrolyzed at the two acyl groups for the generation of the open ring type strongly fluorescent diaminorhodamine, thus the fluorescence response consists of multiple steps, and therefore it has drawbacks such as poor real-time response property and poor suitability for quantification. In order to solve these problems, there has been desired development of a fluorescent probe that can cause a structural change from a closed ring structure to an open ring structure by a reaction at one reaction site to completely achieve off/on control of fluorescence.